Method for analyzing substance

ABSTRACT

A method for analyzing a substance, comprising: a step of obtaining a dispersion complex by co-existing fine particles having a dispersion property higher than that of lyophobic fine particles in a dispersion phase of the lyophobic fine particles at a concentration sufficient for covering a periphery of a lyophobic fine particle group; a step of contacting the dispersion complex and a fluid containing an analyte; and a step of analyzing the analyte using an optical measuring means, wherein the lyophobic fine particles exist in the dispersion phase in a group for providing the surface enhancing effect. The method is effective for analysis of a slight amount substance or a low concentration substance.

TECHNICAL FIELD

[0001] The present invention relates to all the analysis methodutilizing the surface enhancing effect in a vibration spectroscopymethod. In particular, it relates to the life science field, whichrequires analysis of an organism component, such as the functionanalysis of a protein in an aqueous solution, and furthermore, theearth, environment field, which requires analysis of a hazardoussubstance in the environment, or the like.

BACKGROUND TECHNOLOGY

[0002] Clarification of the physiological function of the bio functionalsubstances, such as a protein and a nucleic acid has been an importanttask. In addition thereto, control and design of the physical chemicalproperties are indispensable for realization of a bioreactor, abiosensor, a DNA chip, and for the near further, a bio element, or thelike. Therefore, development of the analysis methods for the physicalchemical properties of the bio functional substances and analysisreagents for the measurement is expected.

[0003] A bio functional substance has the activity in an organism, andthe organism has water as a medium. Therefore, clarification of thephysical chemical properties should be carried out in an aqueoussolution. As an analysis method effective for examining the structure ofa bio functional substance and the interaction with the substratemolecules in water, the Raman spectroscopy as a kind of the vibrationspectroscopy method has been provided.

[0004] However, since the signal intensity obtained by the ordinaryRaman spectroscopy is extremely low and the sensitivity is poor, aspecimen concentration of several % or more has been required.Therefore, in the case of a bio functional substance, a specimencondensing operation is necessary and indispensable so that problems areinvolved in the cost, and the risk of losing or denaturation of thespecimen during the operation.

[0005] In contrast, even in the case of the Raman spectroscopy, if aspecimen interacts with metal fine particles, the surface enhancingeffect of amplifying the signal intensity is known (“Surface enhancedRaman Scattering”, ed. By R. K. Chang and t. E. Furtak, (PlenumPublishing, N.Y., 1982) ). The enhancing sensitivity is said to be ingeneral from 10,000 times to 1,000,000 times.

[0006] The surface enhancing effect appears dramatically in the case theabove-mentioned metal fine particles are used in an aggregated state. Ameasuring method utilizing the surface enhancing effect is importantalso as a study method for the biotechnology (K. Kneipp, H. Kneipp, I.Itzkan, R. R. Dasari, and M. S. Feld, Biomedical Applications of Lasers,77 (7), 915-924 (1999); Surface-enhanced Raman scattering: A new toolfor biomedical spectroscopy).

[0007] According to a recent experiment using aggregation of preciousmetal fine particles, the surface enhancing effect reaching as much as100,000,000,000,000 times capable of one molecule detection has beenconfirmed (K. Kneipp, H. Kneipp, R. Manoharan, E. Hanlon, I. Itzkan, R.R. Dasari, and M. S. Feld, Applied Spectroscopy, 52 (12), 1493-1497(1998); Extremely large enhancement factors in surface-enhanced Ramanscattering for molecules on colloidal gold clusters). Such metal fineparticles are referred to as the substrate of the surface enhancingeffect. Semiconductors such as a gallium and a gallium arsenide can alsobe the substrate.

[0008] As embodiments at the time of using metal fine particles as thesubstrate for the surface enhancing effect, a colloid of metal fineparticles, a film with metal fine particles deposited on the surface inan island-like form, a glass matrix with metal fine particles dispersedin the inside by a sol gel method, a polymer matrix with metal fineparticles dispersed in the inside, or the like has been reported so far.Moreover, the present inventors have also disclosed in the officialgazette of the Japanese Patent Application Laid Open (JP-A) No. 11-61209a technique for obtaining a stable dispersion of the precious metal fineparticles by producing precious metal fine particles by the reducingreaction in a dispersion with plate-like fine particles of a swellablelayered silicate, or the like dispersed.

[0009] Among the substrates, a colloid of nano precious metal fineparticles in an aqueous solution is regarded as most convenience in thepractical use. As the reason therefor, advantages such as 1) capabilityof synthesizing fine particles in a liquid phase method, andfacilitation of handling, 2) adoptability to a continuous flow analysissystem, 3) capability of controlling the particle size and the shape, 4)capability of easily defining the surface area, and 5) capability ofchanging the morphology for the theoretical analysis, have been pointedout (M. Kerker, D. S. Wang, H. Chew. 0. Siiman, and L. A. Bumm, “SurfaceEnhanced Raman Scattering”, ed. by R. K. Chang and T. E. Furtak, (PlenumPublishing, N.Y., 1982), pp. 109-128; Enhanced Raman scattering bymolecules adsorbed at the surface of colloidal particles).

[0010] In any case, in order to use metal fine particles as thesubstrate for the surface enhancing effect, the dispersion state shouldbe maintained stably. Conventionally, as the method for controlling thedispersion state, since the metal fine particles are lyophobic fineparticles, 1) addition of a stabilizing agent in a liquid phase, 2)deposition onto a solid phase (including coating), 3) containment into amatrix of the above-mentioned glass, polymer, or the like, 4)coexistence of a swellable layered silicate with metal fine particles asmentioned in the above-mentioned official gazette of the Japanese PatentApplication Laid Open (JP-A) No. 11-61209, or the like have beenproposed.

[0011] Among these controlling methods, as a stabilizing agent to beused in a liquid phase, a surfactant such as a sodium dodecyl sulfate, apolyvinyl alcohol, a polyvinyl pyridine, a polyethylene glycol, anN-vinyl pyrrolidone, a bovine serum albumin, a γ-globulin, and aprotective colloid such as a gelatin are known. Moreover, the “methodfor preventing colloid aggregation” of the Japanese Patent ApplicationLaid Open (JP-A) No. 09-070527 discloses the stabilizing effect of abuffer agent such as a tris(hydroxymethyl)amino methane, or the like.

[0012] Moreover, as the deposit onto a solid phase, a method ofdepositing fine particles on a glass plate and stopping aggregation at astage of a certain degree is frequently used. According thereto, it ispossible to deposit the nano fine particles synthesized by the liquidphase method on the glass plate, thereby producing aggregations withdifferent sizes and morphologies.

[0013] However, even if a stabilizing agent is used, it has beenextremely difficult to maintain the aggregation state in the liquidwithout precipitation while maintaining the function of the fineparticles in the dispersion phase of the lyophobic fine particles with aliquid used as the dispersion medium such as a colloid of precious metalfine particles in an aqueous solution As a result, the reproductivityand the stability of a production method for a substrate of the surfaceenhancing effect dependent on the aggregation state of the lyophobicfine particles have been poor, and the performance is stillinsufficient.

[0014] Moreover, since the conventional stabilizing agent preventsaggregation by restraining approach of the fine particles with eachother by adhering on the surface of the fine particles, the surfaceactivity, which is an important function of the metal fine particles, islost. Even in the case the metal fine particles are deposited on thesolid phase, the dispersion and the deviation of the aggregation sizedistribution are large so that the reproductivity of the productionmethod is poor, and furthermore, it is instable, and thus the stabilitycan be maintained for about several days at most even if they are coatedwith an organic monomolecular layer such as a thiol. According to thecontainment in a matrix, loss of the surface activity of the metal fineparticles by the matrix, and deterioration of the substance moving ratein the matrix are generated so that a substrate with the excellentsurface enhancing effect cannot be obtained. The technique disclosed inthe official gazette of the Japanese Patent Application Laid Open (JP-A)No. 11-61209 produces metal fine particles in a dispersion withplate-like fine particles dispersed so that a long time is required forthe production of the metal fine particles, resulting in rise of thecost, and furthermore, since an acetone dicarboxylic acid, which caneasily be decomposed, is used as the reducing agent, it is inconvenientin terms of handling.

[0015] That is, a substrate with the surface enhancing effect, beingstable for a long time, capable of providing a quick response, to beproduced with a good productivity while maintaining a high surfaceactivity of the lyophobic fine particles, has not been known so far.

[0016] Therefore, an object of the present invention is to provide ananalysis method of keeping lyophobic fine particles existing in a groupas aggregation to be a dispersed phase, and using the obtaineddispersion complex as the practical substrate of the surface enhancingeffect.

DISCLOSURE OF THE INVENTION

[0017] In order to achieve the above-mentioned object, an analysismethod of the present invention has the following characteristics. Thatis, a dispersion complex is obtained by co-existing fine particleshaving a dispersion property higher than that of the lyophobic fineparticles (hereinafter referred to as the “dispersible fine particles”),in a dispersion phase of lyophobic fine particles existing in a group,at a concentration sufficient for covering the periphery of thelyophobic fine particle group. The dispersion complex accordinglyobtained is used as the substrate of the surface enhancing effect. Then,the dispersion complex and a fluid containing an analyte are contactedfor measuring the concentration of the analyte or the nature by anoptical measuring means while utilizing the surface enhancing effectobtained by the approach of the analyte to the lyophobic fine particlegroup.

[0018] According to the present invention, since the group state of thelyophobic fine particles can be maintained by the dispersible fineparticles, excessive progress of the aggregation and generation of theprecipitation can be restrained. As a result, the performance as thesubstrate of the surface enhancing effect can be maintained over a longtime so that analysis using the surface enhancing effect can be carriedout easily.

DETAILED DISCLOSURE OF THE INVENTION

[0019] A dispersion complex is produced in general by a methodcomprising the following steps (a) to (d) in the present invention.

[0020] (a) A step of dispersing lyophobic fine particles in a liquidphase.

[0021] (b) A step of starting aggregation by adding an aggregatingagent, or the like, for obtaining a group of the lyophobic fineparticles.

[0022] (c) A step of adding dispersible fine particles so as to obtain aconcentration sufficient for covering the periphery of the lyophobicfine particle group to the liquid phase after the step (b).

[0023] (d) A step of collecting the obtained group of the lyophobic fineparticles as a dispersion complex.

[0024] As the lyophobic fine particles, although they are notparticularly limited, metal fine particles containing as the maincomponent at least one selected from the group consisting of gold,silver, copper, platinum, nickel, indium and palladium, which has a 1 to100 nm particle size close to the atom size, or semiconductor fineparticles of gallium or gallium arsenide, or the like can be used.However, it is not limited thereto, and any fine particles capable ofproviding a function different from the bulk can be used.

[0025] The lyophobic fine particles are not particularly limited, andthey can be provided to the above-mentioned (b) step as they are aftersynthesis by a liquid phase method. Moreover, by adding and agitatingfine particles obtained by another method in a liquid, they may bedispersed in the liquid phase.

[0026] The method for starting the aggregation and obtaining thelyophobic fine particle group is not particularly limited, and one ormore of the following five means can be selected: (1) increase of theparticle concentration, (2) the ionic strength rise by addition of anelectrolyte, which brings about the salting out phenomenon, such as asodium chloride and an aluminum sulfate, (3) cross linking by a polymer,(4) the temperature rise, and (5) decrease of the polarity of thedispersion medium.

[0027] The stability of the fine particle dispersion phase in the liquidcan be explained by the DLVO theory. An explanation of &n example ofgold fine particles by the DLVO theory will be provided hereafter, andthe other lyophobic fine particles can be explained in the same manner.Gold fine particles, which have been reduced chemically, adsorb areducing agent anion or a complex metal anion, thereby being chargednegatively (M. A. Hayat Ed. “Colloidal gold” vol. 1 and vol. 2, AcademicPress Inc., 1984). In the case the relative size balance between theelectrostatic repulsion potential and the attraction potential dependenton the Van Der Waals force, or the like is appropriate, a local maximumappears in the total potential curve. Unless the kinetic energy of thefine particles is larger than the local maximum, the fine particlescannot approach to each other beyond the local maximum and aggregationis not generated, so that the system is stabilized. In contrast, in thecase the electrostatic repulsion potential is changed by adsorption ofcounter ions, or the like, or the ionic strength increase, the localmaximum value of the total potential curve is reduced, so that theparticles come to be aggregated beyond the energy barrier. On the otherhand, cross linking aggregation is generated due to the existence of apolymer capable of cross-linking a plurality of fine particles.

[0028] The fine particles having a dispersion property higher than thatof the lyophobic fine particles refer to fine particles having the localmaximum appearing in the total potential curve in the above-mentionedDLVO theory at a position with an inter particle distance wider thanthat shown by the local maximum position of the functional fineparticles, at the start of aggregation. Or the fine particles having adispersion property higher than that of the lyophobic fine particlesrefer to the fine particles having a total potential curve with asufficiently large local maximum appearing at a position showing asufficient inter particle distance, in a state that the local maximum ofthe total potential curve of the lyophobic fine particles is vanishing.Since the shape of the total potential curve is determined mainly by theHamaker constant and the Stern electric potential value, the kinds ofthe lyophobic fine particles are not particularly limited by thedefinition.

[0029] As specific examples of the dispersible fine particles, aswellable layered silicate such as a smectite can be presented.

[0030] The reason why the swellable layered silicate is presentedparticularly as an example of the dispersible fine particles is that theface surface thereof is charged negatively and it continues to dispersesufficiently, even in the situation that the lyophobic fine particlesare aggregated and deposited. Even in the case where it is deposited ina severer situation, it maintains a moderate aggregation form to someextent and it is re-dispersed easily by the shearing force such as theagitation Moreover, it has a small particle size so that the number ofthe particles existing in a dispersion is sufficient even with a slightconcentration by weight, thereby covering the periphery of the lyophobicfine particles. Moreover, since the shape is plate-like, the interparticle interaction can easily be generated so that the apparentviscosity of the dispersion medium is raised, the diffusion speed of theparticles is lowered, and the progress of the aggregation of thelyophobic fine particles can be prevented.

[0031] Either a synthetic one or a natural one can be used as thelayered silicate, and a synthetic one is used preferably. A swellableclay mineral obtained by refining a natural one can also be usedpreferably. Unlike a natural one, since a synthetic one is chemicallyhomogeneous, it contains little impurities, it has a high swellingproperty without containing an aggregating ion, and furthermore, it hasa high transparency without including a color metal such as an ironbetween the layers, it is suitable for an optical measuring means.

[0032] Such a swellable layered silicate is commercially available.

[0033] As the dispersion medium for the dispersion phase in the presentinvention, a liquid or a gas of water, alcohol, a hydrocarbon, or thelike can be used, and it is not particularly limited, but water can beused preferably. At the time, the dispersion complex of the presentinvention is collected as a sol with water as a dispersion medium. Inaddition, the dispersion complex of the present invention is collectedas a gel by means of drying, or the like.

[0034] The concentration of the dispersible fine particles in thepresent invention is not particularly limited as long as it is regardedas being sufficient for covering the periphery of the lyophobic fineparticles to be controlled. Here, the concept of covering the peripherymeans that the number of the fine particles having a higher dispersionproperty exceeds drastically that of the lyophobic fine particlesexisting in a unit space. As a result, it may be sufficient forrestraining progress of aggregation or precipitation of the lyophobicfine particles to be controlled. For example, in the case where thenumber concentration of the lyophobic fine particles is about 12^(th)power of 10 in 1,000 cc, the dispersible fine particles of 15^(th) powerof 10 can be regarded as sufficient. As a specific example, a preferableconcentration of the lyophobic fine particles necessary for execution ofthe analysis is 0.01 mM to 4 M, and the concentration of the dispersiblefine particles necessary for covering the lyophobic fine particles inthe concentration range is in general 0.1 g/L or more in the case of aswellable layered silicate. In contrast, the maximum preparingconcentration of the dispersible fine particles of a swellable layeredsilicate is in general 250 g/L, and 300 g/L in the case of condensation.However, it is not limited thereto, and it depends on the kind and theconcentration of the lyophobic fine particles, the means for startingthe aggregation, and the nature of the dispersible fine particlesthemselves.

[0035] The dispersion state of the lyophobic fine particles can becontrolled by the present invention. An example of controlling andstabilizing the lyophobic fine particles in an assembly state as in thepresent invention has not been known so far. Moreover, an example ofstabilizing for more than one month as in the present invention has notbeen known neither. A dispersion complex of the lyophobic fine particleswith the dispersion state controlled can be obtained by the presentinvention. The dispersion complex has the lyophobic fine particleassembly existing in a state preferable for providing the function, sothat it is applicable to the optical element, the sensor, the catalyst,or the like.

[0036] As the analyte in the present invention, an amino acid, a base, aprotein, and a nucleic acid in an aqueous solution can be presented.Moreover, an aromatic chlorine compound in the environment, or the likecan be presented. However, it is not limited thereto.

[0037] As the optical measuring means in the present invention, thevibration spectroscopy such as the Raman spectroscopy and the infraredspectroscopy can be used. As the optical measuring means utilizing thesurface enhancing effect, the RAS (reflection absorption spectroscopy),the SEWS (surface electromagnetic wave spectroscopy), the SEIRA(surface-enhanced infrared spectroscopy), the SERS (surface-enhancedRaman spectroscopy) the SERRS (surface-enhanced resonance Ramanspectroscopy), the SEHRS (surface-enhanced hyper Raman scattering), orthe like are known.

[0038] In the analysis method of the present invention, as the lyophobicfine particles, metal fine particles of gold, silver, copper, platinum,nickel, indium and palladium, or semiconductor fine particles of galliumor gallium arsenide, which have been confirmed to provide the surfaceenhancing effect in the wavelength range used for the vibrationspectroscopy, can be used preferably. In the analysis method of thepresent invention, as the dispersible fine particles, a syntheticsmectite having a good transparency can be used preferably.

[0039] Since the dispersion complex of the present invention can work asthe substrate of the surface enhancing effect for at least several monthwithout adsorption of the metal fine particles on the pipe wall or thewall of the container in the flow system, analysis can be carried out inthe flow system. In the case the dispersion complex is a sol, it canflow in the flow system as the substrate of the surface enhancingeffect, can be brought into contact with the specimen solutioncontaining the analyte, and can be measured by the optical measuringmeans. As an example of the flow system, a capillary electrophoresis,and various kinds of chromatographies are known.

[0040] Moreover, in the case where the dispersion complex is a gel, itcan be used in contact with the specimen solution containing the analytelike a sensor. Preferably, the dispersion complex can be used as adisposable sensor. Of course the sensor may comprise a part of the flowsystem.

[0041] According to the dispersion complex of the present invention, bymodifying the synthetic smectite with at least one substance selectedfrom the group consisting of a ligand compound, an antibody, an antigen,an enzyme, an enzyme substrate, a nucleic acid, and a complement of anucleic acid, the function of recognizing or orienting the analyte canbe provided. In this case, since the sensitization degree of the surfaceenhancing effect is significantly lowered if the distance from thesurface of the lyophobic fine particles is larger, a recognizedsubstance or a part of the functional groups by the orientation cancontribute to the surface enhancing effect so that it can be utilizedfor the selective measuring method for the substances.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042]FIG. 1 shows absorption spectra of a sol containing a gold fineparticle group and a synthetic smectite;

[0043]FIG. 2 shows absorption spectra of the same sol after 20 days;

[0044]FIG. 3 shows absorption spectra of a sol containing a gold fineparticle group and a montmorillonite;

[0045]FIG. 4 shows absorption spectra of a dry gel containing asynthetic smectite and a gold fine particle group;

[0046]FIG. 5 shows Raman spectra containing a pyridine and a gold fineparticle group;

[0047]FIG. 6 shows the time passage transition of the Raman signalintensity of a sol containing a pyridine and a gold fine particle group;and

[0048]FIG. 7 is an calibration curve of an aqueous solution containing adispersion complex and a pyridine.

BEST MODE FOR CARRYING. OUT THE INVENTION

[0049] Preparation Example of a Dispersion Complex Sol

[0050] Gold fine particles were synthesized in an aqueous solutionaccording to the chemical reduction method of adding a sodium citrate to120 cc of a 0.6 mM aqueous chloroaurate solution so as to be 1.6 mM. Asa result of measurement of the average particle size of the gold fineparticles by the small angle X ray scattering method, it was about 40nm. After measuring the absorbance of the obtained gold fine particlecontaining aqueous solution, it was divided into four containers, and asodium chloride was introduced to each container as the aggregatingagent so that the concentration becomes 50 mM/L and began to cause theaggregation.

[0051] Then, in the first container, the color of the gold fine particleliquid was changed from the original red to the reddish purple, thebluish purple, the reddish brown, the brown and black, and finally itwas precipitated. To the second to fourth containers, a slurry of asynthetic smectite (produced by Laporte Corp.) was added each afterpassage of a predetermined time. Then, the color change was stopped, sothat a dispersion complex showing a different color was obtainedaccording to the synthetic smectite addition period. The passage timesfrom the addition of the sodium chloride to the addition of thesynthetic smectite were set so as to be the second container(hereinafter, the aggregation state A)<the third container (hereinafter,the aggregation state B)<the fourth container (hereinafter, theaggregation state C). FIG. 1 shows the absorption spectra of the statebefore the sodium chloride addition (=before the aggregation), theaggregation state A, the aggregation state B and the aggregation stateC. For the comparison, the absorbance of only the synthetic smectite wasmeasured.

[0052] In the case the particle size is same, since the color of thegold colloid depends on the aggregation state of the particles (N. G.Khlebtsov, V. A. Bogatyrev, L. A. Dykman, and A. G. Melnikov, J. ColloidInterface Sci., 180 (2), 436-445 (1996); Spectral Extinction ofColloidal Gold and Its Biospecific Conjugates.), from the results shownin FIG. 1, it was found that a dispersion complex with the assemblystate of the gold fine particles controlled can be produced according tothe method of the present invention.

[0053] Stability Evaluation 1 of a Dispersion Complex Sol

[0054] The absorption spectrum of the solution in the first container inthe above-mentioned preparation example, and the absorption spectraafter passage of 20 days of the dispersion complexes of the aggregationstates A to C are each shown in FIG. 2. As shown in FIG. 2, it was foundthat the dispersion complex containing the synthetic smectite maintainsthe state with the assembly of the gold fine particles controlled, sothat it is stabilized over a long term.

[0055] Stability Evaluation 2 of a Dispersion Complex Sol

[0056] A dispersion complex was prepared in the same manner as in theaggregation state A in the above-mentioned preparation example exceptthat a montmorillonite (produced by Kunimine Kogyo Corp.) was usedinstead of the synthetic smectite. Although the color change was notobserved for several minutes even if it was left in a room temperature,it was changed gradually to the brown over several days. However,precipitation was not generated. The absorption spectra immediatelyafter the preparation, after one minute, and after passage of 20 daysare shown in FIG. 3. As shown in FIG. 3, it was found that thedispersion complex containing the montmorillonite is stabilized whilemaintaining the state that the assembly of the gold fine particles arecontrolled.

[0057] Preparation Example of a Dispersion Complex Gel and StabilityEvaluation

[0058] Polystyrene plates with the surface processed to be hydrophilicby the ultraviolet ray radiation were prepared so that the dispersioncomplexes of the above-mentioned aggregation states A to C were droppedonto the plates, and dried for gelation. For the comparison, a gold fineparticle-containing solution not containing a synthetic smectite wasdropped onto the plate in the same manner. The gels containing thesynthetic smectite were spread evenly on the plates while maintainingthe reddish brown or brown color. The state was stable for at least sixmonths. In contrast, the gold fine particles derived from the solutionnot containing the synthetic smectite were spread unevenly on the plateas a black sediment. For the confirmation, the absorption spectra of thesolid dispersion complexes obtained on the plates are shown in FIG. 4.As shown in FIG. 4, it was found that it was stabilized whilemaintaining the state that the assembly of the gold fine particles iscontrolled owing to the existence of the synthetic smectite.

EXAMPLE 1

[0059] After preparation of the dispersion complex (aggregation state B)of the above-mentioned sol preparation example, 570 μl thereof wascollected, followed by mixing well with 30 μl of a 0.05 M pyridineaqueous solution, thereby obtaining a first liquid mixture.Independently thereof, 570 μl of pure water was collected, followed bymixing well with 30 μl of a 0.05 M aqueous pyridine solution, therebyobtaining a second liquid mixture. The Raman spectra of the two kinds ofthe liquid mixtures were measured using a Fourier transform infraredspectrophotometer “Nicolet magna 650” comprising a Raman module by a1,064 nm excitation wavelength.

[0060] As a result, the ring breathing vibration (about 1,010 cm⁻¹) of a2.5 mM pyridine was remarkably observed in the first liquid mixture. Incontrast, the pyridine of the concentration was not at all observed inthe second liquid mixture (ordinary Raman spectroscopy). Therefore, itwas found that the above-mentioned dispersion complex can provide thesubstrate of the surface enhancing effect, enabling the easy analysisusing the surface enhancing effect with a commercially available Ramanspectroscopy device.

[0061] According to a silicic acid compound such as a smectite, ingeneral, the Raman peak appears at: 485 cm⁻¹ Si—O—Si rocking/bending ofhydrated silicate 809 cm⁻¹ SiO2 silicate chain mode 976 cm⁻¹ Si—Ostretch of bulk chain,

[0062] however, it didn't appear in the first liquid mixture. Therefore,it was revealed that there is no interference of the silicic acidcompound. This point extremely disagrees with the conventionalbackground report showing the Raman peak derived from a silicic acidcompound in the Raman spectrum of a sol gel glass (F. Akbarian, B. S.Dunn, and J. I. Zink, J. Raman Spectrosc., 27 (10), 775-783 (1996):Porous sol-gel silicates containing gold particles as matrixes forsurface-enhanced Raman spectroscopy).

EXAMPLE 2

[0063] After passage of one day after preparation of the above-mentioneddispersion complex (aggregation state B) of the above-mentioned solpreparation example, 570 μl thereof was collected, followed by mixingwell with 30 μl of a 0.05 M pyridine aqueous solution, thereby obtaininga third liquid mixture. Similarly, after passage of one day afterpreparation of a gold fine particle aggregation liquid not containing asynthetic smectite, 570 μl thereof was collected, followed by mixingwell with 30 μl of a 0.05 M pyridine aqueous solution, thereby obtaininga fourth liquid mixture. Results of measurement of the Raman spectra ofthe two kinds of the liquid mixtures using a Fourier transform infraredspectrophotometer “Nicolet magna 650” comprising a Raman module by a1,064 nm excitation wavelength are shown in FIG. 5.

[0064] As shown in FIG. 5, the ring breathing vibration (about 1,010cm⁻¹) of a 2.5 mM low concentration pyridine was remarkably observed inthe third liquid mixture containing the synthetic smectite. In contrast,it was hardly observed in the fourth liquid mixture not containing thesynthetic smectite. Therefore, it was found that the above-mentioneddispersion complex is a substrate of the surface enhancing effect,enabling also after passage of one day the easy analysis using thesurface enhancing effect with a commercially available Ramanspectroscopy device.

EXAMPLE 3

[0065] The dispersion complex (aggregation state B) of theabove-mentioned sol preparation example was stored at a roomtemperature. After passage of predetermined days, the Raman spectrum ofthe pyridine was measured by the method of the example 1 and, then, thetransition of the ring breathing vibration signal intensity was plottedwith respect to the dispersion complex storage period ( mark in thefigure), thereby obtaining FIG. 6. For the comparison, the fourth liquidmixture of the example 2 (x mark in the figure) was used. As shown inFIG. 6, it was found that the above-mentioned dispersion complex worksas the substrate of the surface enhancing effect over a long term ofalmost two month.

EXAMPLE 4

[0066] Pyridine aqueous solutions having the dispersion complex of thepresent invention as the substrate of the surface enhancing effect wereanalyzed with the concentration of the aqueous pyridine solutionschanged in the same manner as in the example 1. A calibration curve madefrom the analysis results are shown in FIG. 7. From FIG. 7, it was foundthat the dispersion complex according to the present invention works asthe substrate of the surface enhancing effect usable for theconcentration measurement.

INDUSTRIAL APPLICABILITY

[0067] As mentioned above, the substance analysis method of the presentinvention is effective for analysis of a slight amount substance or alow concentration substance.

1. An analysis method for a substance, comprising: a step of obtaining adispersion complex by co-existing fine particles having a dispersionproperty higher than that of lyophobic fine particles in a dispersionphase of the lyophobic fine particles at a concentration sufficient forcovering a periphery of a lyophobic fine particle group; a step ofcontacting the dispersion complex and a fluid containing an analyte; anda step of analyzing the analyte using an optical measuring means,wherein the lyophobic fine particles exist in the dispersion phase in agroup for providing the surface enhancing effect.
 2. The analysis methodaccording to claim 1, wherein the lyophobic fine particles are groupedby the function of an aggregating agent.
 3. The analysis methodaccording to claim 1, wherein the optical measuring means is selectedfrom the group consisting of the Raman spectroscopy and the infraredspectroscopy.
 4. The analysis method according to claim 3, wherein thelyophobic fine particles containing as the main component at least onemetal selected from the group consisting of gold, silver, copper,platinum, nickel, indium and palladium.
 5. The analysis method for asubstance according to claim 4, wherein the fine particles having adispersion property higher than that of the lyophobic fine particles areselected from the group consisting of swellable layered silicates suchas a synthetic smectite.
 6. The analysis method according to claim 1,wherein the property of the dispersion complex is a sol having water asthe main medium, and the concentration of the fine particle having ahigh dispersion property that is sufficient for covering the peripheryof the lyophobic fine particle group is 0.1 g/L or more.
 7. The analysismethod according to claim 1, wherein the property of the dispersioncomplex is a sol having water as the main medium, and the concentrationof the fine particle having a high dispersion property that issufficient for covering the periphery of the lyophobic fine particlegroup is 300 g/L or less.
 8. The analysis method according to claim 1,wherein the property of the dispersion complex is a gel obtained from asol having water as the main medium.
 9. The analysis method according toclaim 1, wherein the system for contacting the dispersion complex with afluid containing the analyte for analyzing the analyte is a flow system.10. The analysis method according to claim 5, wherein the swellablelayered silicate modifies at least one substance selected from the groupconsisting of a ligand compound, an antibody, an antigen, an enzyme, anenzyme substrate, a nucleic acid, and a complement of a nucleic acid.